Methods of differentiating preadipocytes and uses thereof

ABSTRACT

Described herein are methods of transdifferentiating preadipocytes, populations of transdifferentiated preadipocytes, and methods of using the transdifferentiated preadipocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to co-pending U.S.Provisional Patent Application No. 62/218,719, filed on Sep. 15, 2015,entitled “DIRECT CONVERSION OF HUMAN PREADIPOCYTES INTO HEMATOPOIETIC,NEURONAL AND PANCREATIC ALPHACELLS BY OCT4 AND KLF4 OVEREXPRESSION,” thecontents of which is incorporated by reference herein in its entirety.

This application also claims the benefit of and priority to co-pendingU.S. Provisional Patent Application No. 62/294,646, filed on Feb. 12,2016, entitled “METHODS OF DIFFERENTIATING PREADIPOCYTES AND USESTHEREOF,” the contents of which is incorporated by reference herein inits entirety

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled 2222251010_ST25.bd, created on Sep. 15, 2015.The content of the sequence listing is incorporated herein in itsentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows one embodiment of a method of transdifferentiatingpreadipocytes.

FIGS. 2A-2D show micrograph images of colony formation duringtransdifferentiation and reprogramming of human preadipocytes bylentiviral overexpression.

FIG. 3 shows a graph demonstrating principal component analysis on geneexpression data of microarray displays distinct cell populations(preadipocyte, OK; single clone, OK_pool; mixed colonies, AdiPS; iPScell) and clustering of replicates (n=2).

FIG. 4 shows a graph demonstrating relative signal intensity frommicroarray displays no significant changes in pluripotent markers thathighly expressed in the fully reprogrammed AdiPS cells.

FIG. 5 shows a graph demonstrating relative signal intensity frommicroarray displays down regulation of fibroblast markers while theexpression of EMT activators (Slug, N-Cad) maintains highly.

FIG. 6 shows a diagram of BioGPS gene annotation in transdifferentiatedpreadipocytes expressing ectopic Oct4 and Klf4. The number of lineageand tissue specific genes is summarized.

FIG. 7 shows a graph demonstrating relative signal intensity frommicroarray displays upregulation of blood cell specific gene expressionfrom two factor induced OK cell (Single clone) and OK_pool (mixedcolonies).

FIG. 8 shows a graph demonstrating brain specific gene expression intransdifferentiated preadipocytes.

FIG. 9 shows a graph demonstrating pancreatic progenitor and isletspecific gene expression in transdifferentiated preadipocytes.

FIGS. 10A-10O show graphs demonstrating expression of genes GCG (FIG.10A), IRX (FIG. 10B), PCSK1 (FIG. 10C), PDK4 (FIG. 10D), RGS4 (FIG.10E), CNTN1 (FIG. 10F), LPPR4 (FIG. 10G), LOXL2 (FIG. 10H), KCTD12 (FIG.10I), KL (FIG. 10J), ISL1 (FIG. 10K), HDAC9 (FIG. 10L), KCNJ2 (FIG.10M), CD36 (FIG. 10N), FABP4 (FIG. 10O) from transdifferentiatedpreadipocytes.

FIGS. 11A-11D show images demonstrating results of immunocytochemistryof transdifferentiated pancreatic alpha cells expressing ectopic Oct4and Klf4. The Oct4 and Klf4 expressing cells were seeded on matrigelplates and subjected to immunocytochemistry using glucagon (FIGS. 11Aand 11B) and NeuroD (FIGS. 11C and 11D) specific antibodies and DAPInuclear counterstaining.

FIG. 12 shows one embodiment of a method to transdifferentiation andreprogramming of human preadipoctyes to pancreatic alpha cells by Oct4lentiviral overexpression.

FIGS. 13A-13D show micrograph images of colony formation duringtransdifferentiation and reprogramming of human preadipocytes bylentiviral overexpression.

FIGS. 14A-14N show graphs demonstrating gene expression as measured byreal-time PCR of GCG (FIG. 14A), KL (FIG. 14B), LPPR4 (FIG. 14C), PDK4(FIG. 14D), CNTN1 (FIG. 14E), KCTD12 (FIG. 14F), LOXL2 (FIG. 14G), PCSK1(FIG. 14H), IRX2 (FIG. 14I), ISL1 (FIG. 14J), HDAC9 (FIG. 14K), KCNJ2(FIG. 14L), CD36 (FIG. 14M), and FABP4 (FIG. 14N) in Oct4 inducedpancreatic alpha cells (Oct4#1 and Oct4#5; single clones, Oct4, mixedcolonies).

FIGS. 15A-15D show micrograph images demonstrating immunocytochemistryof transdifferentiated pancreatic alpha cells expressing ectopic Oct4.The Oct4 induced pancreatic alpha cells were seeded on matrigel platesand subjected to immunocytochemistry using glucagon (FIGS. 15A-15B) andNeuroD (FIGS. 15C-15D) specific antibodies and DAPI nuclearcounterstaining.

FIGS. 16A-16C show micrograph images of preadipocytes that have beentransdifferentiated down adipogenic (FIG. 16A), chondrogenic (FIG. 16B),and osteogenic (FIG. 16C) lineages.

FIGS. 17A-17D show fluorescent micrograph images of 2 factor (OK) and 4factor (OKSM) transfected cells using highly proliferative, low passagepreadipocytes (<3 passages).

FIG. 18 shows a graph demonstrating the percent of GFP positive cells inthe cell populations shown in FIGS. 17A-17D, which indicates lentiviraltransduction rates.

FIG. 19 shows a graph demonstrating expression of hematopoietictranscription factors (CD45+ cells) in preadipocytes, OK cells, andOK_pool.

FIG. 20 shows a graph demonstrating expression of hematopoietictranscription factors (Oct4+ cells) in preadipocytes, OK cells, andOK_pool.

FIG. 21 shows a graph demonstrating expression of hematopoieticcytokines in preadipocytes, OK cells, and OK_pool.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, biochemistry, botany and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

DEFINITIONS

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within ±10% of the indicated value, whichever is greater.

As used herein, “nucleic acid” and “polynucleotide” generally refer to astring of at least two base-sugar-phosphate combinations and refers to,among others, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.“Polynucleotide” and “nucleic acids” also encompasses such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. For instance, theterm polynucleotide includes DNAs or RNAs as described above thatcontain one or more modified bases. Thus, DNAs or RNAs comprisingunusual bases, such as inosine, or modified bases, such as tritylatedbases, to name just two examples, are polynucleotides as the term isused herein. “Polynucleotide” and “nucleic acids” also includes PNAs(peptide nucleic acids), phosphorothioates, and other variants of thephosphate backbone of native nucleic acids. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, but contain the same bases. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “nucleic acids” or“polynucleotide” as that term is intended herein.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid(RNA)” can refer to any polyribonucleotide or polydeoxribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be inthe form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA(ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNAinterference construct), siRNA (short interfering RNA), or ribozymes.

As used herein, “nucleic acid sequence” and “oligonucleotide” alsoencompasses a nucleic acid and polynucleotide as defined above.

As used herein, “DNA molecule” includes nucleic acids/polynucleotidesthat are made of DNA. Similarly, “RNA molecule” includes nucleicacids/polynucleotides that are made of RNA.

As used herein, “gene” refers to a hereditary unit corresponding to asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a characteristic(s) ortrait(s) in an organism. The term “gene” encompasses specific nucleotidesequences of a genome that are transcribed into an RNA product and arenot translated into a protein as well as those genomic sequences thatare transcribed into an RNA product yet are translated into a protein.

As used herein, the terms “exogenous DNA,” “exogenous nucleic acidsequence,” “exogenous polynucleotide,” “exogenous gene (includingspecifically named genes)” are used interchangeably with the terms“ectopic DNA,” “ectopic nucleic acid sequence,” “ectopicpolynucleotide,” “ectopic gene (including specifically named genes)” andrefer to a nucleic acid sequence that was introduced into a cell,organism, or organelle via transfection. Exogenous nucleic acidsoriginate from an external source, for instance, the exogenous nucleicacid may be from another cell or organism and/or it may be syntheticand/or recombinant. While an exogenous nucleic acid sometimes originatesfrom a different organism or species, it may also originate from thesame species (e.g., an extra copy or recombinant form of a nucleic acidthat is introduced into a cell or organism in addition to or as areplacement for the naturally occurring nucleic acid). Typically, theintroduced exogenous sequence is a recombinant sequence.

As used herein, the term “recombinant” generally refers to anon-naturally occurring nucleic acid, nucleic acid construct, orpolypeptide. Such non-naturally occurring nucleic acids may includenatural nucleic acids that have been modified, for example that havedeletions, substitutions, inversions, insertions, etc., and/orcombinations of nucleic acid sequences of different origin that arejoined using molecular biology technologies (e.g., a nucleic acidsequences encoding a “fusion protein” (e.g., a protein or polypeptideformed from the combination of two different proteins or proteinfragments), the combination of a nucleic acid encoding a polypeptide toa promoter sequence, where the coding sequence and promoter sequence arefrom different sources or otherwise do not typically occur togethernaturally (e.g, a nucleic acid and a constitutive promoter), etc.).Recombinant also refers to the polypeptide encoded by the recombinantnucleic acid. Non-naturally occurring nucleic acids or polypeptidesinclude nucleic acids and polypeptides modified by man.

As used herein, “polypeptides” or “proteins” are as amino acid residuesequences. Those sequences are written left to right in the directionfrom the amino to the carboxy terminus. In accordance with standardnomenclature, amino acid residue sequences are denominated by either athree letter or a single letter code as indicated as follows: Alanine(Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp,D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

As used herein, “plasmid” as used herein refers to a non-chromosomaldouble-stranded DNA sequence including an intact “replicon” such thatthe plasmid is replicated in a host cell.

As used herein, the term “vector” or is used in reference to a vehicleused to introduce an exogenous nucleic acid sequence into a cell. Avector may include a DNA molecule, linear or circular (e.g. plasmids),which includes a segment encoding a polypeptide of interest operativelylinked to additional segments that provide for its transcription andtranslation upon introduction into a host cell or host cell organelles.Such additional segments may include promoter and terminator sequences,and may also include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from yeast or bacterial genomicor plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, “promoter” includes all sequences capable of drivingtranscription of a gene. In particular, the term “promoter” as usedherein can refer to a DNA sequence generally described as the 5′regulator region of a gene, located proximal to the start codon. Thetranscription of an adjacent gene sequence is initiated at the promoterregion. The term “promoter” also includes fragments of a promoter thatare functional in initiating transcription of the gene. The term“promoter” can encompass constitutive promoters and inducible promoters.

As used herein, “constitutive promoter” is a promoter that allows forcontinual or ubiquitous transcription of its associated gene orpolynucleotide. Constitutive promoters are generally are unregulated bycell or tissue type, time, or environment.

As used herein, “inducible promoter” is a promoter that allowstranscription of its associated gene or polynucleotide in response to asubstance or compound (e.g. an antibiotic, or metal), an environmentalcondition (e.g. temperature), developmental stage, or tissue type.

As used herein, “wild-type” is the average form of an organism, variety,strain, gene, protein, or characteristic as it occurs in a givenpopulation in nature, as distinguished from mutant forms that may resultfrom selective breeding, recombinant engineering, and/or transformationwith a transgene.

As used herein, “operatively linked” indicates that the regulatorysequences useful for expression of the coding sequences of a nucleicacid are placed in the nucleic acid molecule in the appropriatepositions relative to the coding sequence so as to effect expression ofthe coding sequence. This same definition is sometimes applied to thearrangement of coding sequences and transcription control elements (e.g.promoters, enhancers, and termination elements), and/or selectablemarkers in an expression vector.

As used herein, “expression” as used herein describes the processundergone by a structural gene to produce an RNA molecule and/orpolypeptide. It can refer to the combination of transcription andtranslation. Expression can refer to the “expression” of a nucleic acidto produce a RNA molecule and can also refers to “expression” of apolypeptide, indicating that the polypeptide is being produced viaexpression of the corresponding nucleic acid.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purposes and included to minimize ordistinguish the effect of variables other than an independent variable.A “control” can be a positive control, a negative control, or an assayor reaction control (an internal control to an assay or reactionincluded to confirm that the assay was functional). In some instances,the positive or negative control can also be the assay or reactioncontrol.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” includes a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term farmanimal includes a horse, sheep, goat, chicken, pig, cow, donkey, llama,alpaca, turkey, and the like.

As used herein, “passage,” “passaging” and the like, is a term of artthat, in the context of cell culture, can refer to the process ofsubculturing a population of cells and includes physically removing asubset of cells from a cell population and expanding the subsetseparately from the original population in a fresh culture environment.As used herein “passaging” does not include simple media changes whereno subset of the original population is isolated and propagated.

As used herein, “expansion” or “expanded” in the context of cells,refers to an increase in the number of a characteristic cell type, orcell types, from an initial population of cells, which may or may not beidentical. The initial cells used for expansion need not be the same asthe cells generated from expansion. For instance, the expanded cells maybe produced by ex vivo or in vitro growth and differentiation of theinitial population of cells. Expansion can also refer to allowing a cellpopulation to undergo one or more cell division without passaging thecells.

As used herein, the abbreviation “Oct4” can refer to octamer-bindingtranscription factor 4 gene, gene product, and/or protein. Oct4 can beinvolved in the self-renewal of undifferentiated embryonic stem cells.One of ordinary skill in the art will instantly appreciate RNA andprotein variants, homologues, orthologues, and analogues of the Oct4 inhumans and animals. For example, the Oct4 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumbers NM_203289, NM_001173531, NM_001285986, NM_001285987.1,NM_002701, NM_001252452, and NM_013633. Other mRNA sequences that can betranslated to form an Oct4 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, Oct4 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession numbersNP_001272916.1, NP_002692.2, NP_001272915.1, and NP_038661.2. Other Oct4polypeptides in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art.

As used herein, the abbreviation “Klf4”, can refer to krupppel-likefactor 4 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the Klf4 in humans and animals. Forexample, the Klf4 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession numbers NM_001314052 andNM_010637. Other mRNA sequences that can be translated to form a Klf4protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, Klf4 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession numbers NP_004226.3,NP_001300981.1, and NP_034767.2. Other Klf4 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein the abbreviation “Sox2” can refer to SRY(sex determiningregion Y)-box 2 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the Sox2 in humans andanimals. For example, the Sox2 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession numbersNM_003106 and NM_011443. Other mRNA sequences that can be translated toform a Sox2 protein or variant thereof in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, Sox2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession numbers NP_003097.1 andNP_035573.3. Other Sox2 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein the abbreviation “cMyc” can refer to the Myc moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the cMyc in humans and animals. Forexample, the cMyc mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession numbers NM_002467 andNM_001177352, NM_001177353, NM_001177354, and NM_010849. Other mRNAsequences that can be translated to form a cMyc protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, cMyc protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession numbers NP_002458.2, NP_001170823.1, NP_001170824.1,and NP_001170825.1. Other cMyc polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “CNTN1” can refer to contactin 1 gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the CNTN1 in humans and animals. For example, the CNTN1mRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession numbers NM_001256063 and NM_001256064,NM_001843, NM_175038, NM_001159647, NM_001159648, and NM_007727. OthermRNA sequences that can be translated to form a CNTN1 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, CNTN1 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession numbers NP_001242992.1, NP_001242993.1, NP_001834.2,NP_778203.1, NP_001153119.1, NP_001153120.1, NP_031753.1. Other CNTN1polypeptides in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art.

As used herein, the abbreviation “PCSK1” can refer to proproteinconvertase 1 gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the PCSK1 in humans andanimals. For example, the PCSK1 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession numbersNM_001177876.1, NM_000439, NM_001177875, and NM_013628. Other mRNAsequences that can be translated to form a PCSK1 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, PCSK1 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession numbers NP_000430.3, NP_001171346.1, and NP_038656.1.Other PCSK1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PDK4” can refer to pyruvatedehydrogenase kinase isozyme 4 gene, gene product, and/or protein. Oneof ordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the PDK4 in humansand animals. For example, the PDK4 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_002612. Other mRNA sequences that can be translated to form a PDK4protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, PDK4 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002603.1.Other PDK4 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGS4” can refer to regulator of Gprotein signaling gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the RGS4 in humans andanimals. For example, the RGS4 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001102445, NM_001113380, NM_001113381, NM_005613, and NM_009062.Other mRNA sequences that can be translated to form a RGS4 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, RGS4 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001095915.1, NP_001106851.1,NP_001106582.1, NP_005604.1, and NP_033088.2. Other RGS4 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “IRX2” can refer to Iroquois-classhomeodomain gene and/or protein gene, gene product, and/or protein. Oneof ordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the IRX2 in humansand animals. For example, the IRX2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001134222.1, NM_033267.4, and NM_010574. Other mRNA sequences thatcan be translated to form an IRX2 protein or variant thereof in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art. Similarly, IRX2 protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_001127694.1, NP_150366.1, and NP_034704.1 Other IRX2polypeptides in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art.

As used herein, the abbreviation “LPPR4” can refer to phospholipidphosphatase related 4 (also known as lipid phosphate phosphatase-relatedprotein type 4) gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the LPPR4 in humans andanimals. For example, the LPPR4 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001166252.1 and NM_014839.4. Other mRNA sequences that can betranslated to form a LPPR4 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, LPPR4 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001159724.1 and NP_055654.2. Other LPPR4 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “LOXL2” can refer to lysyl oxidase like2 gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the LOXL2 in humans and animals. Forexample, the LOXL2 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_002318.2.Other mRNA sequences that can be translated to form a LOXL2 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, LOXL2 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_002309.1. Other LOXL2 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “LOXL4” can refer to lysyl oxidase like4 gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the LOXL4 in humans and animals. Forexample, the LOXL4 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_032211.6.Other mRNA sequences that can be translated to form a LOXL4 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, LOXL4 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_115587.6. Other LOXL4 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “KCTD12” can refer to the potassiumchannel teteramerization domain containing 12 gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of theKCTD12 in humans and animals. For example, the KCTD12 mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_138444.3. Other mRNA sequences that can betranslated to form a KCTD12 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, KCTD12 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_612453.1. Other KCTD12 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KL” can refer to the Klotho gene, geneproduct, and/or protein. One of ordinary skill in the art will instantlyappreciate RNA and protein variants, homologues, orthologues, andanalogues of the KL in humans and animals. For example, the KL mRNA canhave a nucleotide sequence as specified by, but not limited to, any ofGenBank Accession number(s) NM_004795.3 and NM_153683.2. Other mRNAsequences that can be translated to form a KL protein or variant thereofin human or other animal species can be identified and detected by oneof ordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, KL protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_004786.2. Other KL polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “RBPJ” can refer to recombinationsignal binding protein for immunoglobulin kappa J region gene, geneproduct, and/or protein. One of ordinary skill in the art will instantlyappreciate RNA and protein variants, homologues, orthologues, andanalogues of the KL in humans and animals. For example, the KL mRNA canhave a nucleotide sequence as specified by, but not limited to, any ofGenBank Accession number(s) NM_005349.3, NM_015874.4, NM_203283.2, andNM_203284.2. Other mRNA sequences that can be translated to form a KLprotein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, KL protein can have a polypeptide sequences as specified by,but not limited to, any of GenBank Accession number(s) NP_005340.2,NP_056958.3, NP_976028.1, and NP_976029.1. Other KL polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “Sox9” can refer to SRY-Box 9 gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the Sox9 in humans and animals. For example, the Sox9mRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_000346.3. Other mRNA sequencesthat can be translated to form a Sox9 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, Sox9 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_000337.1. Other Sox9 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “NeuroD” can refer to neuronaldifferentiation 1 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the NeuroD in humans andanimals. For example, the NeuroD mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_002500.4. Other mRNA sequences that can be translated to form aNeuroD protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, NeuroD protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002491.2.Other NeuroD polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “ISL1” can refer to ISL LIM Homeobox 1gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the ISL1 in humans and animals. Forexample, the ISL1 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_002202.2.Other mRNA sequences that can be translated to form an ISL1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, ISL1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_002193.2. Other ISL1 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “RGS2” can refer to regulator ofG-Protein Signaling 2 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the RGS2 in humansand animals. For example, the RGS2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_002923.3. Other mRNA sequences that can be translated to form a RGS2protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, RGS2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002914.1.Other RGS2 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IL8” can refer to interleukin 8 (alsoknown as C-X-C motif ligand 8) gene, gene product, and/or protein. Oneof ordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the IL8 in humansand animals. For example, the IL8 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_000584.3. Other mRNA sequences that can be translated to form an IL8protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, IL8 protein can have a polypeptide sequences as specified by,but not limited to, any of GenBank Accession number(s) NP_000575.1.Other IL8 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AKAP7” can refer to A-kinase anchoringprotein 7 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the AKAP7 in humans and animals. Forexample, the AKAP7 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_004842.3,NM_016377.3, and NM_138633.2. Other mRNA sequences that can betranslated to form an AKAP7 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, AKAP7 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_004833.1, NP_057461.2, and NP_619539.1. Other AKAP7 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “CPE” can refer to carboxypeptidase Egene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CPE in humans and animals. Forexample, the CPE mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001873.3.Other mRNA sequences that can be translated to form a CPE protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, CPE protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001864.1. Other CPE polypeptides in humanor other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “OAS1” can refer to2′-5′-Oligoadenylate Synthetase 1 gene, gene product, and/or protein.One of ordinary skill in the art will instantly appreciate RNA andprotein variants, homologues, orthologues, and analogues of the OAS1 inhumans and animals. For example, the OAS1 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_001032409.2, NM_001320151.1, NM_002534.3, and NM_016816.3.Other mRNA sequences that can be translated to form an OAS1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, OAS1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001027581.1, NP_001307080.1, NP_002525.2,and NP_058132.2. Other OAS1 polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “PLTP” can refer to phospholipidtransfer protein gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the PLTP in humans andanimals. For example, the PLTP mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001242920.1, NM_001242921.1, NM_006227.3, and NM_182676.2. Other mRNAsequences that can be translated to form a PLTP protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, PLTP protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001229849.1, NP_001229850.1, NP_006218.1,and NP_872617.1. Other PLTP polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “PGS4” can refer to leucine rich repeatcontaining 49 gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the PGS4 in humans andanimals. For example, the PGS4 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001199017.2, NM_001199018.2, NM_001284357.1, and NM_017691.4. OthermRNA sequences that can be translated to form a PGS4 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, PGS4 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001185946.1, NP_001185947.1,NP_001271286.1, and NP_060161.2. Other PGS4 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “RNF128” can refer to ring fingerprotein 128 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the RNF128 in humans and animals. Forexample, the RNF128 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_024539.3 andNM_194463.1. Other mRNA sequences that can be translated to form aRNF128 protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, RNF128 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_078815.3and NP_919445.1. Other RNF128 polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “BEX1” can refer to brain expressedX-linked 1 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the BEX1 in humans and animals. Forexample, the BEX1 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_018476.3.Other mRNA sequences that can be translated to form a BEX1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, BEX1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_060946.3. Other BEX1 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “HDAC9” can refer to histonedeacetylase 9 gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the HDAC9 in humans andanimals. For example, the HDAC9 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001204144.2, NM_001204145.2, NM_001204146.2, NM_001204147.2, andNM_001204148.2. Other mRNA sequences that can be translated to form aHDAC9 protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, HDAC9 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s)NP_001191073.1, NP_001191074.1, NP_001191075.1, NP_001191076.1,NP_001191077.1, NP_001308797.1, NP_001308798.1, NP_001308799.1,NP_001308800.1, NP_001308801.1, NP_001308802.1, NP_001308803.1,NP_001308804.1, NP_001308805.1, NP_001308806.1, NP_001308807.1,NP_001308808.1, NP_001308813.1, NP_001308814.1, NP_001308815.1,NP_001308816.1, NP_001308817.1, NP_001308818.1, NP_001308819.1,NP_001308820.1, NP_001308822.1, NP_001308823.1, NP_001308824.1,NP_001308825.1, NP_001308826.1, NP_001308827.1, NP_001308828.1,NP_001308829.1, NP_001308830.1, NP_001308831.1, NP_055522.1,NP_478056.1, and NP_848510.1, NP_848512.1. Other HDAC9 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “KCNJ2” cam refer to potassiumvoltage-gated channel subfamily J member 2 gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of theKCNJ2 in humans and animals. For example, the KCNJ2 mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_000891.2. Other mRNA sequences that can betranslated to form a KCNJ2 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, KCNJ2 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_000882.1. Other KCNJ2 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD36” can refer to the CD36 moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD36 in humans and animals. Forexample, the CD36 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_000072.3,NM_001001547.2, NM_001001548.2, NM_001127443.1, NM_001127444.1,NM_001289908.1, NM_001289909.1, and NM_001289911.1. Other mRNA sequencesthat can be translated to form a CD36 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, CD36 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_000063.2, NP_001001547.1, NP_001001548.1,NP_001120915.1, NP_001120916.1, NP_001276837.1, NP_001276838.1, andNP_001276840.1. Other CD36 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FABP4” can refer to the fatty acidbinding protein 4 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the FABP4 in humans andanimals. For example, the FABP4 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001442.2. Other mRNA sequences that can be translated to form a FABP4protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, FABP4 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_001433.1.Other FABP4 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD71” can refer to the transferrinreceptor gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD71 in humans and animals. Forexample, the CD71 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001128148.2,NM_001313965.1, NM_001313966.1, and NM_003234.3. Other mRNA sequencesthat can be translated to form a CD71 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, CD71 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001121620.1, NP_001300894.1,NP_001300895.1, and NP_003225.2. Other CD71 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “CHST2” can refer to the carbohydratesulfotransferase 2 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the CHST2 in humans andanimals. For example, the CHST2 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_004267.4. Other mRNA sequences that can be translated to form a CHST2protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CHST2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_004258.2.Other CHST2 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMPD3” can refer to the adenosinemonophosphate deaminase 3 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the AMPD3 in humansand animals. For example, the AMPD3 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_000480.2 NM_001025389.1 NM_001025390.1 NM_001172430.1 NM_001172431.1.Other mRNA sequences that can be translated to form an AMPD3 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, AMPD3 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_000471.1, NP_001020560.1, NP_001020561.1,NP_001165901.1, and NP_001165902.1. Other AMPD3 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “CD58” can refer to the CD58 moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD58 in humans and animals. Forexample, the CD58 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001144822.1and NM_001779.2. Other mRNA sequences that can be translated to form aCD58 protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CD58 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s)NP_001138294.1 and NP_001770.1. Other CD58 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “MINPP1” can refer to the multipleinositol-polyphosphate phosphatase 1 gene, gene product, and/or protein.One of ordinary skill in the art will instantly appreciate RNA andprotein variants, homologues, orthologues, and analogues of the MINPP1in humans and animals. For example, the MINPP1 mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_001178117.1, NM_001178118.1, and NM_004897.4.Other mRNA sequences that can be translated to form a MINPP1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, MINPP1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001171588.1, NP_001171589.1, andNP_004888.2. Other MINPP1 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IDH2” can refer to the isocitratedehydrogenase (NADP(+))2 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the IDH2 in humansand animals. For example, the IDH2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001289910.1, NM_001290114.1, and NM_002168.3. Other mRNA sequencesthat can be translated to form an IDH2 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, IDH2 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001276839.1, NP_001277043.1, andNP_002159.2. Other IDH2 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD70” can refer to the CD70 Molecule(also referred to as tumor necrosis factor (ligand) superfamily, member70) gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD70 in humans and animals. Forexample, the CD70 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001252.4.Other mRNA sequences that can be translated to form a CD70 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, CD70 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001243.1. Other CD70 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “HLA-DRA” can refer to the majorhistocompatibility complex, Class II, DR alpha gene, gene product,and/or protein. One of ordinary skill in the art will instantlyappreciate RNA and protein variants, homologues, orthologues, andanalogues of the HLA-DRA in humans and animals. For example, the HLA-DRAmRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_019111.4. Other mRNA sequencesthat can be translated to form a HLA-DRA protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, HLA-DRA protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_061984.2. Other HLA-DRA polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “NX1” can refer to the neurexin 1 gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the NX1 in humans and animals. For example, the NX1mRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_001135659.2, NM_001320156.2,NM_001320157.3, NM_004801.5, and NM_138735.4. Other mRNA sequences thatcan be translated to form a NX1 protein or variant thereof in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art. Similarly, NX1 protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_001129131.1, NP_001307085.1, NP_001307086.1, NP_004792.1,and NP_620072.1. Other NX1 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD83” can refer to the CD83 moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD83 in humans and animals. Forexample, the CD83 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001040280.1,NM_001251901.1, and NM_004233.3. Other mRNA sequences that can betranslated to form a CD83 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, CD83 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001035370.1, NP_001238830.1, and NP_004224.1. Other CD83 polypeptidesin human or other animal species can be identified and detected by oneof ordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “CD74” can refer to the CD74 moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD74 in humans and animals. Forexample, the CD74 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001025158.2,NM_001025159.2, and NM_004355.3. Other mRNA sequences that can betranslated to form a CD74 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, CD74 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001020329.1, NP_001020330.1, and NP_004346.1. Other CD74 polypeptidesin human or other animal species can be identified and detected by oneof ordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “IFI44L” can refer to the interferoninduced protein 44 like gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the IFI44L in humansand animals. For example, the IFI44L mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_006820.3. Other mRNA sequences that can be translated to form anIFI44L protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, IFI44L protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_006811.2.Other IFI44L polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “BST2” can refer to the bone marrowstromal cell antigen 2 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the BST2 in humansand animals. For example, the BST2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_004335.3. Other mRNA sequences that can be translated to form a BST2protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, BST2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_004326.1.Other BST2 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “ISG15” can refer to the ISG15ubiquitin-like modifier gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the ISG15 in humansand animals. For example, the ISG15 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_005101.3. Other mRNA sequences that can be translated to form anISG15 protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, ISG15 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_005092.1.Other ISG15 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CLIC6” can refer to the chlorideintracellular channel 6 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the CLIC6 in humansand animals. For example, the CLIC6 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001317009.1 and NM_053277.2. Other mRNA sequences that can betranslated to form a CLIC6 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, CLIC6 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001303938.1 and NP_444507.1. Other CLIC6 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “PBX3” can refer to the pre-B-CellLeukemia Homeobox 3 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the PBX3 in humans andanimals. For example, the PBX3 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001134778.1 and NM_006195.5. Other mRNA sequences that can betranslated to form a PBX3 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, PBX3 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001128250.1 and NP_006186.1. Other PBX3 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “TOX2” can refer to TOX high mobilitygroup box family member 2 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the TOX2 in humansand animals. For example, the TOX2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001098796.1, NM_001098797.1, NM_001098798.1, and NM_032883.2. OthermRNA sequences that can be translated to form a TOX2 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, TOX2 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001092266.1, NP_001092267.1,NP_001092268.1, and NP_116272.1. Other TOX2 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “HCP5” can refer to HLA Complex P5gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the HCP5 in humans and animals. Forexample, the HCP5 RNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NR_040662.1.

As used herein, the abbreviation “RAC2” can refer to RAC2 high mobilitygroup box family member 2 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the RAC2 in humansand animals. For example, the RAC2 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_002872.4. Other mRNA sequences that can be translated to form a RAC2protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, RAC2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002863.1.Other RAC2 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGS1” can refer to Regulator ofG-Protein Signaling 1 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the RGS1 in humansand animals. For example, the RGS1 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_002922.3. Other mRNA sequences that can be translated to form a RGS1protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, RGS1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002913.3.Other RGS1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “STAT4” can refer to Regulator ofG-Protein Signaling 1 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the STAT4 in humansand animals. For example, the STAT4 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001243835.1 and NM_003151.3. Other mRNA sequences that can betranslated to form a STAT4 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, STAT4 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001230764.1 and NP_003142.1. Other STAT4 polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “HLA-DMB” can refer to the MajorHistocompatibility Complex, Class II, DM Beta gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of theHLA-DMB in humans and animals. For example, the HLA-DMB mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_002118.4. Other mRNA sequences that can betranslated to form a HLA-DMB protein or variant thereof in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art. Similarly, HLA-DMB protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_002109.2. Other HLA-DMB polypeptides in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art.

As used herein, the abbreviation “NT5E” can refer to the 5′-NucleotidaseEcto gene, gene product, and/or protein. One of ordinary skill in theart will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the NT5E in humans and animals. Forexample, the NT5E mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001204813.1and NM_002526.3. Other mRNA sequences that can be translated to form aNT5E protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, NT5E protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s)NP_001191742.1 and NP_002517.1. Other NT5E polypeptides in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art.

As used herein, the abbreviation “BTG1” can refer to the B-CellTranslocation Gene 1 gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the BTG1 in humans andanimals. For example, the BTG1 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001731.2. Other mRNA sequences that can be translated to form a BTG1protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, BTG1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_001722.1.Other BTG1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CD14” can refer to the CD14 moleculegene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CD14 in humans and animals. Forexample, the CD14 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_000591.3.Other mRNA sequences that can be translated to form a CD14 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, CD14 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_000582.1. Other CD14 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art

As used herein, the abbreviation “F13A1” can refer to the CoagulationFactor XIII A Chain gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the F13A1 in humans andanimals. For example, the F13A1 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_000129.3. Other mRNA sequences that can be translated to form a F13A1protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, F13A1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_000120.2.Other F13A1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FGL2” can refer to the fibrinogen like2 gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the FGL2 in humans and animals. Forexample, the FGL2 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_006682.2.Other mRNA sequences that can be translated to form a FGL2 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, FGL2 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_006673.1. Other FGL2 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “FOLR3” can refer to the FolateReceptor 3 (Gamma) gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the FOLR3 in humans andanimals. For example, the FOLR3 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_000804.3. Other mRNA sequences that can be translated to form a FOLR3protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, FOLR3 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_000795.2.Other FOLR3 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MX1” can refer to the MX Dynamin likeGTPase 1 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the MX1 in humans and animals. Forexample, the MX1 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001144925.2.Other mRNA sequences that can be translated to form a MX1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, MX1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001138397.1. Other MX1 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “PSENEN” can refer to the PresenilinEnhancer Gamma Secretase Subunit gene, gene product, and/or protein. Oneof ordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the PSENEN in humansand animals. For example, the RPSENEN mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_172341.2. Other mRNA sequences that can be translated toform a RSENEN protein or variant thereof in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart. Similarly, PSENEN protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_758844.1. Other PSENEN polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “IFI6” can refer to the InterferonAlpha Inducible Protein 6 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the IFI6 in humansand animals. For example, the IFI6 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_002038.3. Other mRNA sequences that can be translated to form an IFI6protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, IFI6 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002029.3.Other IFI6 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HS3ST2” can refer to the HeparanSulfate-Glucosamine 3-Sulfotransferase 2 gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of theHS3ST2 in humans and animals. For example, the HS3ST2 mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_006043.1. Other mRNA sequences that can betranslated to form a HS3ST2 protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, HS3ST2 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(sNP_006034.1. Other HS3ST2 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “TAGLN3” can refer to the Transgelin 3gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the TAGLN3 in humans and animals. Forexample, the TAGLN3 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_013259.2.Other mRNA sequences that can be translated to form a TAGLN3 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, TAGLN3 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession numbers NP_037391.2. Other TAGLN3 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “SV2B” can refer to the SynapticVesicle Glycoprotein 2B gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the SV2B in humansand animals. For example, the SV2B mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)and NM_014848.6. Other mRNA sequences that can be translated to form aSV2B protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, SV2B protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_055663.1.Other SV2B polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NCALD” can refer to the NeurocalcinDelta gene, gene product, and/or protein. One of ordinary skill in theart will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the NCALD in humans and animals. Forexample, the NCALD mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001040624.1.Other mRNA sequences that can be translated to form a NCALD protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, NCALD protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001035714.1. Other NCALD polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “ENC1” can refer to theEctodermal-Neural Cortex 1 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the ENC1 in humansand animals. For example, the ENC1 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession numbersNM_003633.3. Other mRNA sequences that can be translated to form an ENC1protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, ENC1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_003624.1.Other ENC1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “COBL” can refer to the Cordon-Bleu WH2Repeat Protein gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the COBL in humans andanimals. For example, the COBL mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001287436.1. Other mRNA sequences that can be translated to form aCOBL protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, COBL protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s)NP_001274365.1. Other COBL polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “EEF1A2” can refer to the EukaryoticTranslation Elongation Factor 1 Alpha 2 gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of theEEF1A2 in humans and animals. For example, the EEF1A2 mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_001958.3. Other mRNA sequences that can betranslated to form an EEF1A2 protein or variant thereof in human orother animal species can be identified and detected by one of ordinaryskill in the art using techniques generally known to one of ordinaryskill in the art. Similarly, EEF1A2 protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_001949.1. Other EEF1A2 polypeptides in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art.

As used herein, the abbreviation “KBTBD11” can refer to the Kelch Repeatand BTB Domain Containing 11 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the KBTBD11 inhumans and animals. For example, the KBTBD11 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_001958.3. Other mRNA sequences that can be translated toform a KBTBD11 protein or variant thereof in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart. Similarly, KBTBD11 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_055682.1. Other KBTBD11 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “RGL1” can refer to the Ral GuanineNucleotide Dissociation Stimulator Like 1 gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of the RGL1in humans and animals. For example, the RGL1 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_015149.4. Other mRNA sequences that can be translated toform a RGL1 protein or variant thereof in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, RGL1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_055964.3.Other RGL1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CA11” can refer to the CarbonicAnhydrase 11 gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the CA11 in humans andanimals. For example, the CA11 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001217.4. Other mRNA sequences that can be translated to form a CA11protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CA11 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_001208.2.Other CA11 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NETO2” can refer to the Neuropilin andTolloid Like 2 gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the NETO2 in humans andanimals. For example, the NETO2 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_018092.4. Other mRNA sequences that can be translated to form a NETO2protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, NETO2 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_060562.3.Other NETO2 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “DOK5” can refer to the Docking Protein5 gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the DOK5 in humans and animals. Forexample, the DOK5 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_018431.4.Other mRNA sequences that can be translated to form a DOK5 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, DOK5 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_060901.2. Other DOK5 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “TUBB2B” can refer to the Tubulin Beta2B Class IIb gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the TUBB2B in humans andanimals. For example, the TUBB2B mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_178012.4. Other mRNA sequences that can be translated to form aTUBB2B protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, TUBB2B protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_821080.1.Other TUBB2B polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “PCDH19” can refer to the Protocadherin19, gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the PCDH19 in humans and animals. Forexample, the PCDH19 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001105243.1.Other mRNA sequences that can be translated to form a PCDH19 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, PCDH19 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001098713.1. Other PCDH19 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “PTPRO” can refer to the ProteinTyrosine Phosphatase, Receptor Type O gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of thePTPRO in humans and animals. For example, the PTPRO mRNA can have anucleotide sequence as specified by, but not limited to, any of GenBankAccession number(s) NM_030667.2. Other mRNA sequences that can betranslated to form a PTPRO protein or variant thereof in human or otheranimal species can be identified and detected by one of ordinary skillin the art using techniques generally known to one of ordinary skill inthe art. Similarly, PTPRO protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_109592.1. Other PTPRO polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “SATB2” can refer to the SATB Homeobox2 gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the SATB2 in humans and animals. Forexample, the SATB2 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_001172509.1.Other mRNA sequences that can be translated to form a SATB2 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, SATB2 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_001165980.1. Other SATB2 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “FGF13” can refer to the FibroblastGrowth Factor 13 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the FGF13 in humans andanimals. For example, the FGF13 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_004114.3. Other mRNA sequences that can be translated to form a FGF13protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, FGF13 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_004105.1.Other FGF13 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CXADR” can refer to the CoxsackieVirus And Adenovirus Receptor gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the CXADR in humansand animals. For example, the CXADR mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_001338.4. Other mRNA sequences that can be translated to form a CXADRprotein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CXADR protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_001329.1.Other CXADR polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MLLT11” can refer to theMyeloid/Lymphoid or Mixed-Lineage Leukemia; Translocated To, 11 gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the MLLT11 in humans and animals. For example, theMLLT11 mRNA can have a nucleotide sequence as specified by, but notlimited to, any of GenBank Accession number(s) NM_006818.3. Other mRNAsequences that can be translated to form a MLLT11 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, MLLT11 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_006809.1. Other MLLT11 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “TMEFF2” can refer to the TransmembraneProtein with EGF Like and Two Follistatin like Domains 2 gene, geneproduct, and/or protein. One of ordinary skill in the art will instantlyappreciate RNA and protein variants, homologues, orthologues, andanalogues of the TMEFF2 in humans and animals. For example, the TMEFF2mRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_016192.3. Other mRNA sequencesthat can be translated to form a TMEFF2 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, TMEFF2 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_057276.2. Other TMEFF2 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “BRSK1” can refer to the BRSerine/Threonine Kinase 1 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the BRSK1 in humansand animals. For example, the BRSK1 mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_032430.1. Other mRNA sequences that can be translated to form a BRSK1protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, BRSK1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_115806.1.Other BRSK1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “NRGN” can refer to the Neurograningene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the NRGN in humans and animals. Forexample, the NRGN mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_006176.2.Other mRNA sequences that can be translated to form a NRGN protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, NRGN protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_006167.1. Other NRGN polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “TAC1” can refer to the TachykininPrecursor 1 gene, gene product, and/or protein. One of ordinary skill inthe art will instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the TAC1 in humans and animals. Forexample, the TAC1 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_003182.2.Other mRNA sequences that can be translated to form a TAC1 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, TAC1 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_003173.1. Other TAC1 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “CDH10” can refer to the Cadherin 10gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the CDH10 in humans and animals. Forexample, the CDH10 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_006727.4.Other mRNA sequences that can be translated to form a CDH10 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, CDH10 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_006718.2. Other CDH10 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “TPD52L1” can refer to the TumorProtein D52-Like 1 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the TPD52L1 in humans andanimals. For example, the TPD52L1 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_003287.3. Other mRNA sequences that can be translated to form aTPD52L1 protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, TPD52L1 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_003278.1.Other TPD52L1 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “HIST2H2BE” can refer to the HistoneCluster 2, H2be gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the HIST2H2BE in humans andanimals. For example, the HIST2H2BE mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_003528.2. Other mRNA sequences that can be translated to form aHIST2H2BE protein or variant thereof in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, HIST2H2BE protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_003519.1. Other HIST2H2BE polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “SLC16A9” can refer to the SoluteCarrier Family 16 Member 9 gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the SLC16A9 inhumans and animals. For example, the SLC16A9 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_194298.2. Other mRNA sequences that can be translated toform a SLC16A9 protein or variant thereof in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart. Similarly, SLC16A9 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_919274.1. Other SLC16A9 polypeptides in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “MEGF10” can refer to the Multiple EGFLike Domains 10 gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the MEGF10 in humans andanimals. For example, the MEGF10 mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_032446.2. Other mRNA sequences that can be translated to form aMEGF10 protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, MEGF10 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_115822.1.Other MEGF10 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “FMNL2” can refer to the Formin like 2gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the FMNL2 in humans and animals. Forexample, the FMNL2 mRNA can have a nucleotide sequence as specified by,but not limited to, any of GenBank Accession number(s) NM_052905.3.Other mRNA sequences that can be translated to form a FMNL2 protein orvariant thereof in human or other animal species can be identified anddetected by one of ordinary skill in the art using techniques generallyknown to one of ordinary skill in the art. Similarly, FMNL2 protein canhave a polypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_443137.2. Other FMNL2 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “KIAA1598” can refer to the Shootin 1gene, gene product, and/or protein. One of ordinary skill in the artwill instantly appreciate RNA and protein variants, homologues,orthologues, and analogues of the KIAA1598 in humans and animals. Forexample, the KIAA1598 mRNA can have a nucleotide sequence as specifiedby, but not limited to, any of GenBank Accession number(s)NM_001127211.2. Other mRNA sequences that can be translated to form aKIAA1598 protein or variant thereof in human or other animal species canbe identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, KIAA1598 protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)NP_001120683.1. Other KIAA1598 polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “DNER” can refer to the Delta/NotchLike EGF Repeat Containing gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the DNER in humansand animals. For example, the DNER mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_139072.3. Other mRNA sequences that can be translated to form a DNERprotein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, DNER protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_620711.3.Other DNER polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “KAL1” can refer to the Anosmin 1 gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the KAL1 in humans and animals. For example, the KAL1mRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_000216.3. Other mRNA sequencesthat can be translated to form a KAL1 protein or variant thereof inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, KAL1 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_000207.2. Other KAL1 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “CD45” can refer to the ProteinTyrosine Phosphatase, Receptor Type C gene, gene product, and/orprotein. One of ordinary skill in the art will instantly appreciate RNAand protein variants, homologues, orthologues, and analogues of the CD45in humans and animals. For example, the CD45 mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) NM_002838.4. Other mRNA sequences that can be translated toform a CD45 protein or variant thereof in human or other animal speciescan be identified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CD45 protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_002829.3.Other CD45 polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “AMY1C” can refer to the Amylase, Alpha1C (Salivary) gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the AMY1C in humans andanimals. For example, the AMY1C mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_001008219.1. Other mRNA sequences that can be translated to form anAMY1C protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, AMY1C protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s)NP_001008220.1. Other AMY1C polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “AMY1A” can refer to the Amylase, Alpha1A (Salivary) gene, gene product, and/or protein. One of ordinary skillin the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the AMY1A in humans andanimals. For example, the AMY1A mRNA can have a nucleotide sequence asspecified by, but not limited to, any of GenBank Accession number(s)NM_004038.3. Other mRNA sequences that can be translated to form anAMY1A protein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, AMY1A protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_004029.2.Other AMY1A polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “GCG” can refer to the glucagon gene,gene product, and/or protein. One of ordinary skill in the art willinstantly appreciate RNA and protein variants, homologues, orthologues,and analogues of the GCG in humans and animals. For example, the GCGmRNA can have a nucleotide sequence as specified by, but not limited to,any of GenBank Accession number(s) NM_002054.4. Other mRNA sequencesthat can be translated to form a GCG protein or variant thereof in humanor other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art. Similarly, GCG protein can have a polypeptidesequences as specified by, but not limited to, any of GenBank Accessionnumber(s) NP_002045.1. Other GCG polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

As used herein, the abbreviation “CEBPD” can refer to the CCAAT/EnhancerBinding Protein Delta gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the CEBPD in humansand animals. For example, the CEBPD mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_005195.3. Other mRNA sequences that can be translated to form a CEBPDprotein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CEBPD protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_005186.2.Other CEBPD polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “CEBPD” can refer to the CCAAT/EnhancerBinding Protein Delta gene, gene product, and/or protein. One ofordinary skill in the art will instantly appreciate RNA and proteinvariants, homologues, orthologues, and analogues of the CEBPD in humansand animals. For example, the CEBPD mRNA can have a nucleotide sequenceas specified by, but not limited to, any of GenBank Accession number(s)NM_005195.3. Other mRNA sequences that can be translated to form a CEBPDprotein or variant thereof in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.Similarly, CEBPD protein can have a polypeptide sequences as specifiedby, but not limited to, any of GenBank Accession number(s) NP_005186.2.Other CEBPD polypeptides in human or other animal species can beidentified and detected by one of ordinary skill in the art usingtechniques generally known to one of ordinary skill in the art.

As used herein, the abbreviation “STEAP3” can refer to theSix-Transmembrane Epithelial Antigen of Prostate 3 gene, gene product,and/or protein. One of ordinary skill in the art will instantlyappreciate RNA and protein variants, homologues, orthologues, andanalogues of the STEAP3 in humans and animals. For example, theSTEAP3mRNA can have a nucleotide sequence as specified by, but notlimited to, any of GenBank Accession number(s) NM_182915.2. Other mRNAsequences that can be translated to form a STEAP3 protein or variantthereof in human or other animal species can be identified and detectedby one of ordinary skill in the art using techniques generally known toone of ordinary skill in the art. Similarly, STEAP3 protein can have apolypeptide sequences as specified by, but not limited to, any ofGenBank Accession number(s) NP_878919.2. Other STEAP3 polypeptides inhuman or other animal species can be identified and detected by one ofordinary skill in the art using techniques generally known to one ofordinary skill in the art.

As used herein, the abbreviation “AMT1B” can refer to the ammoniumtransport protein 1B gene, gene product, and/or protein. One of ordinaryskill in the art will instantly appreciate RNA and protein variants,homologues, orthologues, and analogues of the AMT1B in bacteria, fungi,humans, and animals. For example, the AMT1B mRNA can have a nucleotidesequence as specified by, but not limited to, any of GenBank Accessionnumber(s) XM_001692619.1. Other mRNA sequences that can be translated toform an AMT1B protein or variant thereof in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart. Similarly, AMT1B protein can have a polypeptide sequences asspecified by, but not limited to, any of GenBank Accession number(s)XM_001692619.1. Other AMT1B polypeptides in human or other animalspecies can be identified and detected by one of ordinary skill in theart using techniques generally known to one of ordinary skill in theart.

SEQ ID NOs: for the various accession numbers specifically providedherein can be found in Table 1. Other variants of the sequences thatcorrespond with the genes, gene products, and proteins provided hereinwill be appreciated by those of ordinary skill in the art and are withinthe scope of this disclosure.

DISCUSSION

In most tissues, including the pancreas, a mature cell performing aspecialized function represents a terminally differentiated cell that isrestricted in potential. In other words, most mature cells possess theability to carry out specific functions but lack the ability toreplicate and expand the pool of cells. In contrast, progenitor cellstypically lack very specific functionality but have the capacity andplasticity to proliferate and expand a pool of cells. In some instancesit is desirable, particularly for the development of in vitro culturesystems for cells to exhibit specific functionalities attributed to aterminally differentiated cell yet continue to proliferate to allow forculture in vitro.

It was once believed that differentiation was a one-way fate. However,it is now known that it is possible to reprogram differentiated cells tocell having more plasticity that can be directed down a differentdifferentiation pathway. Pancreatic cells (including alpha and betacells) are derived from a common progenitor. Many examples exist of fateconversion between endocrine cell types. Further exocrine cells,particularly acinar cells, have been used to convert the exocrine cellsinto an endocrine lineage, specifically pancreatic beta cells.

A major, yet poorly understood, feature of type 2 diabetes is theexcessive hepatic glucose production and the corresponding insulinresistance leading to fasting hyperglycemia. Recent work hasdemonstrated an increased daily and unregulated plasma glucagon inconcentration in type 2 diabetics. Glucagon secretion by pancreaticalpha-cells is an immediate response to glucopenia. Abnormal secretionof glucagon and other counterregulatory hormones is a hallmark of types1 and 2 diabetes and a major limitation to the use of stronghypoglycemia agents. As such there exists a need for improved diabetestreatments, of which some may target the pancreatic alpha cells anddysregulation of glucagon secretion.

With that said, described herein are methods of transdifferentiatingpreadipocytes into, inter alia, pancreatic cells (e.g. pancreatic alphacells), populations of transdifferentiated preadipocytes, and assaysusing the transdifferentiated preadipocytes to evaluate the effect ofcandidate compounds on a characteristic of the transdifferentiatedpreadipocyte.

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Methods of Transdifferentiating Preadipocytes

Described herein are methods of transdifferentiating preadipocytes.Preadipocytes can be differentiated into hematopoietic, neuronal, and/orpancreatic cells. In some embodiments, the preadipocytes can betransdifferentiated into pancreatic alpha cells. In some embodiments,preadipocytes can be transdifferentiated by overexpressing Oct4 in thepreadipocytes. In some embodiments, the preadipocytes can betransdifferentiated by overexpressing Oct4 with at least one otherfactor from the group of Klf4, Sox2, and cMyc. In some embodiments, thepreadipocytes can be transdifferentiated by overexpressing Oct4 andKlf4. In some embodiments, the preadipocytes can be transdifferentiatedby overexpressing Oct4, Klf4, and Sox2. In some embodiments, thepreadipocytes can be transdifferentiated by overexpressing Oct4, Klf4,Sox2, and cMyc. In embodiments where Oct4 is overexpressed in thepreadipocyte with at least one other factor from the group of Klf4,Sox2, and cMyc, the additional factor(s) can be overexpressed at thesame (simultaneously) or at different times than the Oct4. In someembodiments, preadipocytes can be transdifferentiated withoutoverexpressing Klf4, Sox2, and/or cMyc. Overexpression of Oct4,

In some embodiments, preadipocytes can be transdifferentiated byoverexpressing Oct 4, Klf4, Sox2, and/or cMyc in the preadipocytesfollowed by culturing the cells until about 100% confluent, after which,the cells can be passaged and a subset of cells can be reseeded onto afeeder cell layer. The cells can then be expanded on the feeder layeruntil the desired differentiated cell type is achieved.

During the first culture period, cells can be cultured in a preadipocytegrowth medium. In some embodiments the preadipocyte growth medium cancontain Dulbecco's modified Eagle medium (DMEM) supplemented with FBS atabout 10% and suitable antibiotics. In embodiments, after passaging,cells or subset thereof can be reseeded and expanded on the feeder layerin a suitable stem cell culture medium, such as, but not limited to,mTeSR1 media. In some embodiments, the first culture period can beanywhere from about 1-7 or more days. In other words, the cells can becultured after transfection or transduction for about 1-7 days withoutpassaging. In some embodiments, the cells can be expanded on the feederlayer for about 1-10 days.

Oct4, Klf4, Sox2, and/or cMyc can be ectopically (or exogenously)expressed or overexpressed in preadipocytes by any suitable method.Suitable methods include, but are not limited to viral-based andnon-viral based transduction and transfection techniques. Viral basedtechniques can include those that involve adenoviral, lentiviral, andretroviral systems. Non-virus based techniques include liposomal andmicelle delivery reagents. In some embodiments, the transgene can beincorporated into the genome utilizing CRISPR or other genomemodification technique known by those of skill in the art. In someembodiments, expression or overexpression of Oct4, Klf4, Sox2, and/orcMyc can be transient or stable. In some embodiments, expression oroverexpression of Oct4, Klf4, Sox2, and/or cMyc can be inducible. Thepreadipocytes can be transfected or transduced 1, 2, 3, 4, or more timeswith the same or different transgene(s).

Preadipocytes can be harvested from adipose tissue. In some embodiments,the preadipocytes can be obtained from the stromal vascular fraction ofadipose tissue. In some embodiments the preadipocytes that aretransfected or transduced are low passage preadipocytes. As used herein,“low passage” can refer to cells that have undergone 3 or less passages.

Transdifferentiated Cells

Also described herein are cells derived from transdifferentiated frompreadipocytes according to the methods described herein. Insofar asthese cells are transdifferentiated by at least exogenous expression ofOct4, these cells are not identical to preadipocytes or differentiatedcells even if there are similarities in function or structure to anative or natural cell, such as an isolated preadipocyte or pancreaticcell, and thus not a product of nature.

In some embodiments, the transdifferentiated cells (also referred toherein as transdifferentiated preadipocytes) can have ectopic expressionof Oct4. The transdifferentiated preadipocytes can also have ectopicexpression of Klf4, Sox2, and/or cMyc. In some embodiments thetransdifferentiated preadipocytes only have ectopic expression of Oct4.In further embodiments, the transdifferentiated preadipocytes only haveectopic expression of Oct4 and Klf4. Stated differently, in someembodiments, the transdifferentiated preadipocytes having ectopicexpression of Oct4 do not have ectopic expression of Klf4, Sox2, and/orcMyc.

While transdifferentiated preadipocytes can be similar in function,genotype, phenotype, and/or structure to native pancreatic cells (e.g.pancreatic alpha or beta cells), neuronal cells, and/or hematopoieticcells, the populations of cells provided herein are not native ornatural “isolated” pancreatic cells, neuronal cells, and/orhematopoietic cells insofar as they can have differing expressionpatterns (or profiles) of genes and/or proteins as described hereinand/or have exogenous expression of one or more genes and/or proteins(e.g. Oct4) as described herein. These non-natural cells can be referredto herein as pancreatic cells, neuronal cells, and hematopoietic cells.

In some embodiments the transdifferentiated preadipocytes can expressNeuroD protein. In this way, the transdifferentiated preadipocytes canbe similar to pancreatic alpha cells. In some embodiments, thetransdifferentiated preadipocytes have increased expression of apancreatic alpha cell specific gene as compared to an unmodifiedpreadipocyte. The pancreatic alpha cell specific gene that can controlpancreatic hormone expression and processing can be any one of GCG,CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2, KCTD12, KL, and anycombination thereof. In some embodiments, the transdifferentiatedpreadipocytes have increased expression of a pancreatic cell specificgene as compared to an unmodified preadipocyte. The pancreaticprogenitor cell specific gene can be any one of RBPJ, Sox9, NeuroD,ISL1, RGS2, IL8, KCTD12, AKAP7, PCSK1, LOXL4, CPE, OAS1, PLTP, RNF128,BEX1, CEBPD, HIST2H2BE, HDAC9, KCNJ2, and combinations thereof. In someembodiments, the transdifferentiated preadipocytes can have reduced RNAexpression of CD36 that is a marker for adipocyte stem cells as comparedto an unmodified preadipocyte. In further embodiments, thetransdifferentiated preadipocyte can have reduced RNA expression ofFABP4 that is highly expressed in adipocytes manner as compared to anunmodified preadipocyte.

The transdifferentiated preadipocytes can have increased expression of ahematopoietic cell specific gene as compared to an unmodifiedpreadipocyte cell. In some embodiments, the hematopoietic cell specificgene can be any one of CD71⁺, CHST2, AMPD3, CD58, STEAP3, MINPP1, IDH2,CD70, HLA-DRA, NX1, CD83, CD74, IFI44L, BST2, ISG15, CLIC6, PBX3, TOX2,HOPS, RAC2, RGS1, STAT4, HLA-DMB, NT5E, BTG1, CD14⁺, F13A1, FGL2, FOLR3,MX1, PSENEN, RGS1, and combinations thereof.

The transdifferentiated preadipocytes can have increased expression of aneural cell specific gene as compared to an unmodified preadipocyte. Insome embodiments, the hematopoietic cell specific gene can be any one ofIFI6, HS3ST2, TAGLN3, SV2B, NCALD, ENC1, COBL, EEF1A2, KBTBD11, RGL1,CA11, NETO2, DOK5, TUBB2B, PCDH19, PTPRO, SATB2, FGF13, CXADR, LPPR4,MLLT11, TMEFF2, BRSK1, NRGN, TAC1, PCDH19, CDH10, TPD52L1, HIST2H2BE,SLC16A9, MEGF10, FMNL2, EEF1A2, KIAA1598, DNER, CALB2, KAL1, andcombinations thereof.

In some embodiments, the transdifferentiated cells can be provided in acell culture vessel including flasks, tubes, and multi-well plates. Thetransdifferentiated cells can be provided in a cell culture vessel atany stage of transdifferentiation. In some embodiments, the cells can beseeded on a feeder layer in a cell culture vessel or on anothersubstrate such as an extracellular matrix (synthetic or natural)composition. Other suitable substrates will be appreciated by those ofskill in the art. In some embodiments, the cell culture vessel containsone or more trans-wells.

Uses of the Transdifferentiated Cells

The transdifferentiated preadipocytes described herein can be used totest compounds for their effect on a characteristic of thetransdifferentiated preadipocytes. In some embodiments, the testcompounds are candidate pharmaceutical compounds. In short, thetransdifferentiated preadipocytes can be used in a drug or compoundscreening assay. As described herein are methods of evaluating acandidate compound including the step of contacting a population oftransdifferentiated cells, such as pancreatic alpha cells, with anamount of a candidate compound. In some embodiments, cell growth,pancreatic cell development and differentiation, glucagon production,apoptosis, cytotoxicity, pancreatic cell replacement therapy aremeasured after contacting a population of transdifferentiatedpreadipocytes with a test compound.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1 Introduction

Overexpression of defined transcription factors can convert one celltype into another and has important implications for regenerativemedicine. Ectopic expression of key transcription factors in somaticdonor cells has been used to generate many different cell types,including cells resembling blood cells [1, 2], brown fat cells [3],hepatocytes [4], sertoli cells [5], and various cell types of the neurallineage [6-9].

Expression of Oct4, partially in conjunction with Klf4, is involved inmaintaining pluripotency at early developmental stages including in theblastomere, inner cell mass and epiblast. Oct4 and Klf4 have beenreported as components of four defined transcription factors (Oct4,Sox2, Klf4, cMyc) that trigger reprogramming of somatic cells intopluripotent stem cells (iPS cell) that are functionally identical toembryonic stem cells (ESCs) [10]. Ectopic expression of Oct4 alone, orectopic expression of Oct4 and Klf4, in combination with small moleculetreatment, can be sufficient to generate iPS cells from neural stemcells, mouse embryonic fibroblasts (MEF) and dermal papilla cells[11-14]. Oct4 activates early epithelial-to-mesenchymal transition(EMT), which is involved in somatic cell reprogramming [15]. Szabo andcolleagues observed direct conversion of human dermal fibroblasts intomultipotent blood progenitors by ectopic expression of Oct4 [16]. Thissuggests that Oct4 can function as a haematopoietic lineagedifferentiation marker as well. This Example demonstrates that Oct4 andKlf4 can induce transdifferentiation of human preadipocytes into threedifferent lineages including hematopoietic, neuronal and pancreaticendocrine cells. Furthermore, ectopic expression of Oct4 alone caninduce transdifferentiation of preadipocytes into glucagon expressingpancreatic α cells.

Results

Ectopic Expression of Oct4 and Klf4 can Induce Transdifferentiation ofHuman Preadipocytes.

Primary cultures of preadipocytes from stromal vascular fraction (SVF)of adipose tissues are a rich source of CD34 and alkaline phosphatasepositive adult mesenchymal stem cells [17]. Preadipocytes candifferentiate toward mesodermal lineages including osteocyte, adipocyteand chondrocyte (FIGS. 16A-16C). Two factor (OK) and 4 factor (OKSM)transfected cells were generated using highly proliferative, low passagepreadipocytes (<3 passages) that had been demonstrated to haveapproximately 50% transfection yields using a lentiviral vector (FIGS.17A-17D and 18). FIGS. 1 and 2A-2D show the general strategy. One daybefore lentiviral infection, cells were seeded on 6 well plates at aconcentration of 10⁵ cells per well with Dulbecco's Modified Eagle'smedium (DMEM) containing 10% FBS. For the lentiviral infection, total 25multiplicity of infection (MOI) of lentivirus which expresses Oct4, Klf4under control of CMV promoter (SBI and Cellomics) or polycistroniclentivirus STEMCCA which express four iPS factors (Oct4, Sox2, Klf4,cMyc) were added to the medium with 5 μg/ml polybrene (Sigma-Aldrich)(FIGS. 1 and 2A-2D).

The lentiviral infection was repeated the next day, and the culturemedium was changed with fresh DMEM/5% FBS medium every other day. Whencells became confluent, they were trypsinized, counted and seeded on MEFfeeder layer at a density of 5×10⁴ cells/ml with mTeSR1 medium (StemCellTechnology). Emerging of round cells with colony formation was observedas early as 5 days post feeder layer cultures. The two factor (OK)transfected cells showed similar morphological changes when compared tofour iPS factor (OSKM) transfected cells that were undergoing earlystages of somatic cell reprogramming (FIGS. 1 and 2A-2D) with theexception that more dense colony formation and immortalization from theOSKM overexpressed iPS colonies (AdiPS cells) was observed. Similarmorphological changes have been shown from multi-lineage bloodprogenitors through overexpression of Oct4 in human adult dermal andneonatal foreskin fibroblasts [16]. Nine OK cell lines and multiple iPScell lines were established from OSKM overexpressed preadipocytes bycolony picking.

To further characterize OK transfected cells, global gene expressionpatterns were examined using microarray analysis (Illumina). Samplerelations based on the expression of 20,909 genes showed that OK cells(single clone, mixed colonies) are distinct from preadipocyte and fourfactor (OSKM) induced iPS cells (AdiPS) (FIG. 3). A majority of thepluripotency genes that were highly expressed in the fully reprogrammedAdiPS cells were expressed at lower levels in two factor induced OKcells, but at levels similar to or less than levels observed inpreadipocytes (FIG. 4). Expression of fibroblast and preadipocyte markergenes were expressed at lower levels in OK cells with the exception EMTactivators (Slug, N-Cad) which maintained higher expression levels (FIG.5). In contrast to a previous report [15], this suggested that OK cellsunderwent an epithelial-to-mesenchymal transition (EMT) rather thanreprogramming processes. Epigenetic modifiers including inhibitors forhistone deacetylase (HDAC), histone methyltransferase (HMT) and DNAmethyltransferase (DNMT) are known to facilitate the reprogrammingprocess by targeting epigenetic barriers [18-20]. Small moleculecombination treatments (e.g., BIX-01294, BayK8644, RG-108) have beenreported to enable two factor (Oct4 and Klf4) induced reprogramming ofmouse embryonic fibroblasts [18]. In this Example, exposure to the DNMT1inhibitor Zebularine did not affect reprogramming or global geneexpression patterns (data not shown).

Transdifferentiation of Hematopoietic and Neuronal Lineages by Oct4 andKlf4 Overexpression.

Using a BioGPS gene annotation portal (www.biogps.org), we analyzedlineage and tissue specific expression profiles of the top 292 genescharacterized by a minimum 4 fold-change induced by Oct4 and Klf4overexpression. As summarized in FIG. 6, enrichments of specific genesfrom three germ layers (mesoderm, ectoderm and endoderm) and inner cellmass (ICM) were observed. Overall, 41% of selected genes are annotatedas tissue specific expression while the remaining is ubiquitouslyexpressed. Specifically, a total of 45 genes are designatedhematopoietic cell specific, 59 genes are brain and peripheral nervespecific, and 27 genes are exclusively expressed from pancreas andpancreatic islets. Oct4 can also function as a hematopoietictranscription factor that enhances the expression of panleukocyte markerCD45 in human fibroblasts [16]. Oct4-derived fibroblasts (CD45+ FibOct4)can differentiate into mature blood cells by additional cytokinetreatments. Unlike the Oct4-derived fibroblasts (FibOct4, CD45+FibOct4), OK cells were not observed to alter expression ofhematopoietic transcription factors and cytokines (FIGS. 19-21).However, OK cells were observed to have upregulation of CD71+, earlyerythroid marker genes (CHST2, AMPD3, CD58, STEAP3, MINPP1, IDH2),lymphoblasts marker genes (CD70, HLA-DRA, MX1, CD83, CD74, IFI44L, BST2,ISG15, CLIC6, PBX3, TOX2, HCP5, RAC2, RGS1), lymphocyte marker genes(STAT4, HLA-DMB, NT5E, HCP5, RAC2, BTG1) and CD14+ monocyte/dendriticcell marker genes (F13A1, FGL2, FOLR3, MX1, PSENEN, RGS1) (FIG. 7).

Direct conversion of mouse fibroblasts into neurons by the combinatorialexpression of neural lineage specific transcription factors (Ascl1,Brn2, Myt1l) has been demonstrated [6]. In addition, four reprogrammingfactor (Oct4, Sox2, Klf4, cMyc) induced intermediate cells have beenidentified as neural progenitor cells [21]. Tissue-specific geneexpression pattern analysis revealed that OK cells expressed increasedlevels of brain specific markers (IFI6, HS3ST2, TAGLN3, SV2B, NCALD,ENC1, COBL, EEF1A2, KBTBD11, RGL1, CA11). Some genes are known to beexclusively expressed from fetal brain (NETO2, DOK5, TUBB2B, PCDH19,PTPRO, SATB2, FGF13, CXADR, LPPR4, MLLT11) or regions specific to theamygdala (TMEFF2, BRSK1, NRGN, TAC1P, CDH19), pineal glands (CDH10,TPD52L1, HIST2H2BE, NCALD, CXADR), prefrontal and cortex (SLC16A9, NRGN,MEGF10, FMNL2, EEF1A2, KIAA1598, KBTBD11, RGL1, CA11, SATB2),hypothalamus and thalamus (DNER, CALB2, FMNL2, KIAA1598), and spinalcord (KAL1, DNER, MEGF10). Most of the brain specific genes that werehighly induced in OK cells were undetectable or at very low levels whencompared to untransfected human preadipocytes (FIG. 8). Neuronaldifferentiation, however, did not proceed any further: OK cells did notshow neuronal morphological changes such as neuronal rosettes or nervefibers (axon) (data not shown). In addition, the expression of neurallineage-specific transcription factors (Ascl1, Brn2, Myt1l) were notaltered in OK cells. Without being bound by theory, these resultssuggest that Oct4 and Klf4 induced neural transdifferentiation can beindependent of neural development, and that Oct4 and Klf4 overexpressiondirectly convert mesoderm to ectoderm lineages.

Transdifferentiation of Pancreatic α Cells by Oct4/Klf4 or Oct4 Alone.

As a part of an endocrine organ, pancreatic α and β cells play vitalroles maintaining blood glucose homeostasis by secreting glucagon andinsulin, respectively. Three of pancreatic lineage specifictransdifferentiation factors (Pdx1, Ngn3, Mafa) have been identifiedthat efficiently convert adult hepatocytes and pancreatic exocrine cellsinto insulin secreting pancreatic β cells [22-25]. However, pancreatic αcell specific transdifferentiation has not been reported. Global geneexpression analysis indicated that 27 pancreatic islet specific geneswere enriched in OK cells including pancreatic progenitor markers (RBPJ,Sox9, NeuroD and ISL1) (FIG. 9). The enriched genes are functionallyassociated with G protein related cell signaling and which areresponsible for glucose sensing (RGS2, 1L8, KCTD12, AKAP7),transcription regulation (ISL1, BEX1, CEBPD, HIST2H2BE, NEUROD, SOX9,RBPJ) and enzymes for pancreatic hormone processing (PCSK1, LOXL4, CPE,OAS1, PLTP, RNF128). Among the four pancreatic hormones that secret fromdistinct cells in the pancreatic islets, upregulation of glucagon (GCG)gene (FIG. 9) was observed, but not insulin, somatostatin, or pancreaticpolypeptide (data not shown). Without being bound by theory, theseresults suggest that human preadipocytes can be directly converted topancreatic α cells by Oct4 and Klf4 overexpression.

Pancreatic cell specific gene expression has been confirmed withreal-time RTPCR using SYBR green conjugated gene specific primers thatshow distinct expression patterns across three types of pancreatic cells(α, β and exocrine cells)[26]. As shown in FIGS. 10A-10O, the expressionof pancreatic α cell markers (GCG, CNTN1, PCSK1, PDK4, RGS4, IRX2,LPPR4, LOXL2, KCTD12, KL) and β cell markers (ISL1, HDAC9, KCNJ2) werebeen upregulated dramatically in OK cells while the preadipocyte markergene (CD36, FABP4) expression decreased (FIGS. 10A-10O). The expressionof pancreatic exocrine cell specific markers (AMY1C, AMT1B, AMY1A) werehighly expressed in all samples (data not shown). In order to assessglucagon and NeuroD protein expression (a pancreatic α cell product anda pancreatic progenitor marker, respectively), OK cells were seeded ontomatrigel coated cell culture plates and cells were examined byimmunocytochemistry. The results demonstrate that all of the colonies onmatrigel stained with specific antibodies against glucagon or NeuroDexcept for the feeder cells (FIGS. 11A-11D). To evaluate the secretionof glucagon from the transdifferentiated pancreatic α cells (OK cells,Oct4 alone) using an ELISA from the cell culture supernatants. Althoughthe expression of glucagon from the cell extracts was detected, glucagonsecretion was barely detectable. Without being bound by theory, thissuggests that pancreatic α cells are missing a component for theglucagon processing and secretion while the cells highly expressespancreatic exocrine cell markers such as AMY1C, AMT1B, AMY1A.

Analysis of the regulatory transcription factor binding sites indicatedthat the promoter region of most genes that were upregulated in OK cellscontain putative POU domain binding sequences (5′-ATTTGCAT-3′), but notKlf4 binding sequences, suggesting Klf4 might be dispensable for thetransdifferentiation processes. To test this hypothesis, preadipocyteswere transfected with Oct4 only using a lentiviral vector and repeatedthe experiment using the same conditions used previously. The generaloutline is shown in FIGS. 12 and 13A-13D. Similar to the previous twofactor overexpression, colony formation was observed at 5 days afterculturing on feeder cell layer. The cells had virtually identicalmorphology as the two factor (Oct4, Klf4) induced OK cells (FIGS. 12 and13A-13D). Next, the pancreatic cell specific gene and protein expressionfrom established colonies (Oct4#1, Oct4#5) and mixed colonies (Oct4) wasanalyzed. As shown in FIGS. 14A-14N, the expression of pancreatic cellmarkers from Oct4 single factor overexpressed cells was upregulated atlevels similar to two factor (Oct4, Klf4) induced OK cells, except forLOXL2, RGS4 and PCSK1. Glucagon and NeuroD protein expression wasconfirmed by immunohistochemistry (FIGS. 15A-15D). Without being boundby theory, these results suggest that ectopic expression of Oct4 aloneis sufficient to induce transdifferentiation of human preadipocytes intoglucagon expressing pancreatic α cells. These results are consistentwith previous findings that Oct4 and Nanog play roles in maintainingpluripotency while Klf4 and cMyc regulate cellular division [27].

This Example also indicates that Klf4 is dispensable in thetransdifferentiation processes. The Oct4 induced pancreatic α cells wasexpanded for more than 10 passages without any loss of cellularproliferation properties or pancreatic marker gene expression (data notshown). Glucagon producing pancreatic α cells comprise 0.2-0.5% ofpancreatic islets and show great plasticity to become insulin secretingβ cells after severe β cell loss in mouse model[28]. Furthermore,Bramswig and colleagues demonstrated that histone methyltransferaseinhibitor treatment facilitates the conversion of pancreatic α cell toinsulin secreting β cells[26]. These findings regarding the plasticityof adipose stem cells and transdifferentiation into pancreatic endocrinecells and the development of a simple and efficient method to producepancreatic α cells will provide a model to advance therapeuticstrategies for metabolic disease.

Methods

Cell Culture and Lentiviral Transfection.

Human preadipocytes (LaCell, LA) were maintained with Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% FBS, 50 U/mlpenicillin and 50 μg/ml streptomycin at 37° C., 5% CO2 in a humidifiedincubator. High titer lentivirus, overexpressing human Oct4, Klf4 orpolycistronic lentivirus which express four iPS factors (STEMCCA), waspurchased from System Biosciences (San Diego, Calif.), Cellomics(Halethorpe, Md.) and EMD Millipore (Temecula, Calif.). The day beforelentiviral transfection, human preadipocytes were trypsinized, counted,and seeded in 6-well plates at a density of 10⁵ cells/well. The nextday, culture medium was replaced with pre-warmed medium containing 5μg/ml polybrene (Sigma-Aldrich) and 25 MOI of lentivirus. Lentiviralinfection was repeated the next day, and culture medium was changed withfresh preadipocyte growth medium. When the cells reached confluence,cells were trypsinized, counted and seeded on a prepared MEF feederlayer at a density of 5×10⁴ cells with mTeSR1 medium (StemCellTechnology).

Microarray and Gene Expression Analysis.

Total RNA was prepared from cultures using Trizol Reagent (LifeTechnology) and the RNeasy Mini RNA isolation kit (Qiagen, Valencia,Calif.) with DNase I digestion. The RNA quality was verified using anAgilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, Calif.) usingthe RNA 6000 Pico Assay. Generation of double-stranded cDNA, preparationand labeling of cRNA, hybridization to HumanHT-12 v4 Expression BeadChip(Illumina, San Diego, Calif.), washing, and scanning were all performedaccording to the standard Illumina protocol. Quantitative PCR to measuremRNA expression levels was performed with PrimeTime qPCR assays(Integrated DNA Technologies, Coralville, Iowa) using a 7700 real-timePCR system in the genomic core facility at Pennington BiomedicalResearch Center. Expression levels were compared to known standardsamples and were normalized GAPDH.

Immunocytochemistry.

Cells were fixed with 4% paraformaldehyde in PBS for 10 min, andincubated for 1 hr with antibodies specific for glucagon and NeuroD1(Abcam). After washing three times with PBS, cells were incubated for 1hr with fluorescent conjugated secondary antibody (Invitrogen). Nucleiwere detected by DAPI staining (Vector shield).

Abbreviations Used in the Examples

AdiPS=Adipose derived iPS cellDNMT=DNA methyltransferaseEMT=epithelial-to-mesenchymal transitionESCs=embryonic stem cellsHDAC=histone deacetylaseHMT=histone methyltransferaseICM=inner cell massiPS cell=induced pluripotent stem cellMEF=mouse embryonic fibroblasts

OK=Oct4, Klf4

OKSM=Oct4, Klf4, Sox2, cMycSVF=stromal vascular fraction

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TABLE 1 SEQ ID GenBank NO Accession Number Gene Name  1 NM_203289 OCT4 2 NM_001173531 OCT4  3 NM_001285986 OCT4  4 NM_001285987.1 OCT4  5NM_002701 OCT4  6 NM_001252452 OCT4  7 NM_013633 OCT4  8 NP_001272916.1OCT4  9 NP_002692.2 OCT4  10 NP_001272915.1 OCT4  11 NP_038661.2 OCT4 12 NM_001314052 Klf4  13 NM_010637 Klf4  14 NP_004226.3 Klf4  15NP_001300981.1 Klf4  16 NP_034767.2 Klf4  17 NM_003106 Sox2  18NM_011443 Sox2  19 NP_003097.1 Sox2  20 NP_035573.3 Sox2  21 NM_002467cMyc  22 NM_001177352 cMyc  23 NM_001177353 cMyc  24 NM_001177354 cMyc 25 NM_010849 cMyc  26 NP_002458.2 cMyc  27 NP_001170823.1 cMyc  28NP_001170824.1 cMyc  29 NP_001170825.1 cMyc  30 NM_001256063 CNTN1  31NM_001256064 CNTN1  32 NM_001843 CNTN1  33 NM_175038 CNTN1  34NM_001159647 CNTN1  35 NM_001159648 CNTN1  36 NP_001242992.1 CNTN1  37NP_001242993.1 CNTN1  38 NP_001834.2 CNTN1  39 NP_778203.1 CNTN1  40NP_001153119.1 CNTN1  41 NP_001153120.1 CNTN1  42 NP_031753.1 CNTN1  43NM_001177876.1 PCSK1  44 NM_000439 PCSK1  45 NM_001177875 PCSK1  46NM_013628 PCSK1  47 NP_000430.3 PCSK1  48 NP_001171346.1 PCSK1  49NP_038656.1 PCSK1  50 NM_002612 PDK4  51 NP_002603.1 PDK4  52NM_001102445 RGS4  53 NM_001113380 RGS4  54 NM_001113381 RGS4  55NM_005613 RGS4  56 NM_009062 RGS4  57 NP_001095915.1 RGS4  58NP_001106851.1 RGS4  59 NP_001106582.1 RGS4  60 NP_005604.1 RGS4  61NP_033088.2 RGS4  62 NM_001134222.1 IRX2  63 NM_033267.4 IRX2  64NM_010574 IRX2  65 NP_001127694.1 IRX2  66 NP_150366.1 IRX2  67NP_034704.1 IRX2  68 NM_001166252.1 LPPR4  69 NM_014839.4 LPPR4  70NP_001159724.1 LPPR4  71 NP_055654.2 LPPR4  72 NM_002318.2 LOXL2  73NP_002309.1 LOXL2  74 NM_032211.6 LOXL4  75 NP_115587.6 LOXL4  76NM_138444.3 KCTD12  77 NP_612453.1 KCTD12  78 NM_004795.3 KL  79NP_004786.2 KL  80 NM_005349.3 RBPJ  81 NM_015874.4 RBPJ  82 NM_203283.2RBPJ  83 NM_203284.2 RBPJ  84 NP_005340.2 RBPJ  85 NP_056958.3 RBPJ  86NP_976028.1 RBPJ  87 NP_976029.1 RBPJ  88 NM_000346.3 Sox9  89NP_000337.1 Sox9  90 NM_002500.4 NeuroD  91 NP_002491.2 NeuroD  92NM_002202.2 ISL1  93 NP_002193.2 ISL1  94 NM_002923.3 RGS2  95NP_002914.1 RGS2  96 NM_000584.3 IL8  97 NP_000575.1 IL8  98 NM_004842.3AKAP7  99 NM_016377.3 AKAP7 100 NM_138633.2 AKAP7 101 NP_004833.1 AKAP7102 NP_057461.2 AKAP7 103 NP_619539.1 AKAP7 104 NM_001873.3 CPE 105NP_001864.1 CPE 106 NM_001032409.2 OAS1 107 NM_001320151.1 OAS1 108NM_002534.3 OAS1 109 NM_016816.3 OAS1 110 NP_001027581.1 OAS1 111NP_001307080.1 OAS1 112 NP_002525.2 OAS1 113 NP_058132.2 OAS1 114NM_001242920.1 PLTP 115 NM_001242921.1 PLTP 116 NM_006227.3 PLTP 117NM_182676.2 PLTP 118 NP_001229849.1 PLTP 119 NP_001229850.1 PLTP 120NP_006218.1 PLTP 121 NP_872617.1 PLTP 122 NM_001199017.2 PGS4 123NM_001199018.2 PGS4 124 NM_001284357.1 PGS4 125 NM_017691.4 PGS4 126NP_001185946.1 PGS4 127 NP_001185947.1 PGS4 128 NP_001271286.1 PGS4 129NP_060161.2 PGS4 130 NM_024539.3 RNF128 131 NM_194463.1 RNF128 132NP_078815.3 RNF128 133 NP_919445.1 RNF128 134 NM_018476.3 BEX1 135NP_060946.3 BEX1 136 NM_001204144.2 HDAC9 137 NM_001204145.2 HDAC9 138NM_001204146.2 HDAC9 139 NM_001204147.2 HDAC9 140 NM_001204148.2 HDAC9141 NP_001191073.1 HDAC9 142 NP_001191074.1 HDAC9 143 NP_001191075.1HDAC9 144 NP_001191076.1 HDAC9 145 NP_001191077.1 HDAC9 146NP_001308797.1 HDAC9 147 NP_001308798.1 HDAC9 148 NP_001308799.1 HDAC9149 NP_001308800.1 HDAC9 150 NP_001308801.1 HDAC9 151 NP_001308802.1HDAC9 152 NP_001308803.1 HDAC9 153 NP_001308804.1 HDAC9 154NP_001308805.1 HDAC9 155 NP_001308806.1 HDAC9 156 NP_001308807.1 HDAC9157 NP_001308808.1 HDAC9 158 NP_001308813.1 HDAC9 159 NP_001308814.1HDAC9 160 NP_001308815.1 HDAC9 161 NP_001308816.1 HDAC9 162NP_001308817.1 HDAC9 163 NP_001308818.1 HDAC9 164 NP_001308819.1 HDAC9165 NP_001308820.1 HDAC9 166 NP_001308822.1 HDAC9 167 NP_001308823.1HDAC9 168 NP_001308824.1 HDAC9 169 NP_001308825.1 HDAC9 170NP_001308826.1 HDAC9 171 NP_001308827.1 HDAC9 172 NP_001308828.1 HDAC9173 NP_001308829.1 HDAC9 174 NP_001308830.1 HDAC9 175 NP_001308831.1HDAC9 176 NP_055522.1 HDAC9 177 NP_478056.1 HDAC9 178 NP_848510.1 HDAC9179 NP_848512.1 HDAC9 180 NM_000891.2 KCNJ2 181 NP_000882.1 KCNJ2 182NM_000072.3 CD36 183 NM_001001547.2 CD36 184 NM_001001548.2 CD36 185NM_001127443.1 CD36 186 NM_001127444.1 CD36 187 NM_001289908.1 CD36 188NM_001289909.1 CD36 189 NM_001289911.1 CD36 190 NP_000063.2 CD36 191NP_001001547.1 CD36 192 NP_001001548.1 CD36 193 NP_001120915.1 CD36 194NP_001120916.1 CD36 195 NP_001276837.1 CD36 196 NP_001276838.1 CD36 197NP_001276840.1 CD36 198 NM_001442.2 FABP4 199 NP_001433.1 FABP4 200NM_001128148.2 CD71 201 NM_001313965.1 CD71 202 NM_001313966.1 CD71 203NM_003234.3 CD71 204 NP_001121620.1 CD71 205 NP_001300894.1 CD71 206NP_001300895.1 CD71 207 NP_003225.2 CD71 208 NM_004267.4 CHST2 209NP_004258.2 CHST2 210 NM_000480.2 AMPD3 211 NM_001025389.1 AMPD3 212NM_001025390.1 AMPD3 213 NM_001172430.1 AMPD3 214 NM_001172431.1 AMPD3215 NP_000471.1 AMPD3 216 NP_001020560.1 AMPD3 217 NP_001020561.1 AMPD3218 NP_001165901.1 AMPD3 219 NP_001165902.1 AMPD3 220 NM_001144822.1CD58 221 NM_001779.2 CD58 222 NP_001138294.1 CD58 223 NP_001770.1 CD58224 NM_001178117.1 MINPP1 225 NM_001178118.1 MINPP1 226 NM_004897.4MINPP1 227 NP_001171588.1 MINPP1 228 NP_001171589.1 MINPP1 229NP_004888.2 MINPP1 230 NM_001289910.1 IDH2 231 NM_001290114.1 IDH2 232NM_002168.3 IDH2 233 NP_001276839.1 IDH2 234 NP_001277043.1 IDH2 235NP_002159.2 IDH2 236 NM_001252.4 CD70 237 NP_001243.1 CD70 238NM_019111.4 HLA-DRA 239 NP_061984.2 HLA-DRA 240 NM_001135659.2 NX1 241NM_001320156.2 NX1 242 NM_001320157.3 NX1 243 NM_004801.5 NX1 244NM_138735.4 NX1 245 NP_001129131.1 NX1 246 NP_001307085.1 NX1 247NP_001307086.1 NX1 248 NP_004792.1 NX1 249 NP_620072.1 NX1 250NM_001040280.1 CD83 251 NM_001251901.1 CD83 252 NM_004233.3 CD83 253NP_001035370.1 CD83 254 NP_001238830.1 CD83 255 NP_004224.1 CD83 256NM_001025158.2 CD74 257 NM_001025159.2 CD74 258 NM_004355.3 CD74 259NP_001020329.1 CD74 260 NP_001020330.1 CD74 261 NP_004346.1 CD74 262NM_006820.3 IFI44L 263 NP_006811.2 IFI44L 264 NM_004335.3 BST2 265NP_004326.1 BST2 266 NM_005101.3 ISG15 267 NP_005092.1 ISG15 268NM_001317009.1 CLIC6 269 NM_053277.2 CLIC6 270 NP_001303938.1 CLIC6 271NP_444507.1 CLIC6 272 NM_001134778.1 PBX3 273 NM_006195.5 PBX3 274NP_001128250.1 PBX3 275 NP_006186.1 PBX3 276 NM_001098796.1 TOX2 277NM_001098797.1 TOX2 278 NM_001098798.1 TOX2 279 NM_032883.2 TOX2 280NP_001092266.1 TOX2 281 NP_001092267.1 TOX2 282 NP_001092268.1 TOX2 283NP_116272.1 TOX2 284 NR_040662.1 HCP5 285 NM_002872.4 RAC2 286NP_002863.1 RAC2 287 NM_002922.3 RGS1 288 NP_002913.3 RGS1 289NM_001243835.1 STAT4 290 NM_003151.3 STAT4 291 NP_001230764.1 STAT4 292NP_003142.1 STAT4 293 NM_002118.4 HLA-DMB 294 NP_002109.2 HLA-DMB 295NM_001204813.1 NT5E 296 NM_002526.3 NT5E 297 NP_001191742.1 NT5E 298NP_002517.1 NT5E 299 NM_001731.2 BTG1 300 NP_001722.1 BTG1 301NM_000591.3 CD14 302 NP_000582.1 CD14 303 NM_000129.3 F13A1 304NP_000120.2 F13A1 305 NM_006682.2 FGL2 306 NP_006673.1 FGL2 307NM_000804.3 FOLR3 308 NP_000795.2 FOLR3 309 NM_001144925.2 MX1 310NP_001138397.1 MX1 311 NM_172341.2 PSENEN 312 NP_758844.1 PSENEN 313NM_002038.3 IFI6 314 NP_002029.3 IFI6 315 NM_006043.1 HS3ST2 316NP_006034.1 HS3ST2 317 NM_013259.2 TAGLN3 318 NP_037391.2 TAGLN3 319NM_014848.6 SV2B 320 NP_055663.1 SV2B 321 NM_001040624.1 NCALD 322NP_001035714.1 NCALD 322 NP_001035714.1 NCALD 323 NM_003633.3 ENC1 324NP_003624.1 ENC1 325 NM_001287436.1 COBL 326 NP_001274365.1 COBL 327NM_001958.3 EEF1A2 328 NP_001949.1 EEF1A2 329 NM_001958.3 KBTBD11 330NP_055682.1 KBTBD11 331 NM_015149.4 RGL1 332 NP_055964.3 RGL1 333NM_001217.4 CA11 334 NP_001208.2 CA11 335 NM_018092.4 NETO2 336NP_060562.3 NETO2 337 NM_018431.4 DOK5 338 NP_060901.2 DOK5 339NM_178012.4 TUBB2B 340 NP_821080.1 TUBB2B 341 NM_001105243.1 PCDH19 342NP_001098713.1 PCDH19 343 NM_030667.2 PTPRO 344 NP_109592.1 PTPRO 345NM_001172509.1 SATB2 346 NP_001165980.1 SATB2 347 NM_004114.3 FGF13 348NP_004105.1 FGF13 349 NM_001338.4 CXADR 350 NP_001329.1 CXADR 351NM_006818.3 MLLT11 352 NP_006809.1 MLLT11 353 NM_016192.3 TMEFF2 354NP_057276.2 TMEFF2 355 NM_032430.1 BRSK1 356 NP_115806.1 BRSK1 357NM_006176.2 NRGN 358 NP_006167.1 NRGN 359 NM_003182.2 TAC1 360NP_003173.1 TAC1 361 NM_006727.4 CDH10 362 NP_006718.2 CDH10 363NM_003287.3 TPD52L1 364 NP_003278.1 TPD52L1 365 NM_003528.2 HIST2H2BE366 NP_003519.1 HIST2H2BE 367 NM_194298.2 SLC16A9 368 NP_919274.1SLC16A9 369 NM_032446.2 MEGF10 370 NP_115822.1 MEGF10 371 NM_052905.3FMNL2 372 NP_443137.2 FMNL2 373 NM_001127211.2 KIAA1598 374NP_001120683.1 KIAA1598 375 NM_139072.3 DNER 376 NP_620711.3 DNER 377NM_000216.3 KAL1 378 NP_000207.2 KAL1 379 NM_002838.4 CD45 380NP_002829.3 CD45 381 NM_001008219.1 AMY1C 382 NP_001008220.1 AMY1C 383NM_004038.3 AMY1A 384 NP_004029.2 AMY1A 385 NM_002054.4 GCG 386NP_002045.1 GCG 387 NM_005195.3 CEBPD 388 NP_005186.2 CEBPD 389NM_182915.2 STEAP3 390 NP_878919.2 STEAP3 391 XM_001692619.1 AMT1B 392XM_001692619.1 AMT1B

We claim:
 1. A method of transdifferentiating a preadipocyte, the methodcomprising: overexpressing Oct4 in a preadipocyte; culturing cellsoverexpressing Oct4 until about 100% confluent; passaging the cellsoverexpressing Oct4 after reacting 100% confluency; and expanding cellsoverexpressing Oct 4 on a feeder cell layer.
 2. The method of claim 1,further comprising overexpressing Klf4 in the preadipocyte.
 3. Themethod of claim 2, wherein Klf4 is simultaneously overexpressed withOct4.
 4. The method of claim 1, wherein overexpression of Oct4 occursvia transfection or transduction of the preadipocyte with an exogenousOct4 gene.
 5. The method of claim 1, wherein overexpression of Klf4occurs via transfection or transduction of the preadipocyte with anexogenous Klf4.
 6. The method of claim 1, further comprisingoverexpressing Sox2 in the preadipocyte.
 7. The method of claim 6,wherein overexpression of Sox2 occurs via transfection or transductionof the preadipocyte with an exogenous Sox2.
 8. The method of claim 1,further comprising overexpressing cMyc in the preadipocyte.
 9. Themethod of claim 9, wherein cMyc is simultaneously overexpressed with Oct4.
 10. The method of claim 1, wherein the preadipocytes have beenpassaged 3 times or less prior to overexpression of Oct4, Klf4, Sox2, orcMyc.
 11. The method of claim 1, wherein the cells overexpressing Oct4are expanded on the feeder layer for about 3, 4, 5, 6, 7, 8 or moredays.
 12. The method of claim 1, wherein the cells overexpressing Oct 4are cultured without passaging for about 1, 2, 3, 4, or 5 days prior tothe steps of passaging and expanding.
 13. The method of claim 1, furthercomprising the step of contacting the cells overexpressing Oct 4 with atest compound.
 14. A population of cells comprising: atransdifferentiated preadipocyte having ectopic expression of Oct4,wherein the transdifferentiated preadipocyte produces glucagon, andwherein the transdifferentiated preadipocyte produces NeuroD protein.15. The population of cells of claim 14, wherein the transdifferentiatedpreadipocyte has increased expression of a pancreatic alpha cellspecific gene as compared to an unmodified preadipocyte, wherein thepancreatic alpha cell specific gene is selected from the groupconsisting of: GCG, CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2,KCTD12, KL and any combination thereof.
 16. The population of cells ofclaim 14, wherein the transdifferentiated preadipocyte has increasedexpression of a pancreatic cell specific gene as compared to anunmodified preadipocyte, wherein the pancreatic cell specific gene isselected from the group consisting of: RBPJ, Sox9, NeuroD, ISL1, RGS2,IL8, KCTD12, AKAP7, PCSK1, LOXL4, CPE, OAS1, PLTP, RNF128, BEX1, CEBPD,HIST2H2BE, HDAC9, KCNJ2, and combinations thereof.
 17. The population ofcells of claim 14, wherein the transdifferentiated preadipocyte hasreduced RNA expression of CD36 as compared to an unmodifiedpreadipocyte.
 18. The population of cells of claim 14, wherein thetransdifferentiated preadipocyte has ectopic expression of Klf4.
 19. Amethod of evaluating a candidate compound, the method comprising:contacting a population of cells with an amount of the candidatecompound, wherein the population of cells comprises atransdifferentiated preadipocyte having ectopic expression of Oct4,wherein the transdifferentiated preadipocyte produces glucagon, andwherein the transdifferentiated preadipocyte produces NeuroD protein;and evaluating a characteristic of the population of cells.
 20. Themethod of claim 30, wherein the characteristic is cell growth, celldevelopment, differentiation, glucagon production, apoptosis, orcytotoxicity.